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ISSN 2409-4943. Ukr. Biochem. J., 2019, Vol. 91, N 1

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doi: https://doi.org/10.15407/ubj91.01.005

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© 2019 Varbanets L. D. et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

UDC 579.841.11.22

Рantoea agglomerans lipopolysaccharides:structure, functional and biological activity

L. D. VarBaNetS, Т. V. Bulyhina, l. А. Pasichnyk, n. V. ZhytkeVich

Zabolotny institute of Microbiology and Virology,national academy of sciences of ukraine, kyiv;

e-mail: [email protected]

received: 02 September 2018; accepted: 13 December 2018

this review analyzed literature data, as well as our own research on lipopolysaccharides (lPs) of gram-negative bacteria, focusing mainly on Pantoea agglomerans, a member of the enterobacteriaceae fami-ly. the unique structures of O-specific polysaccharide chains of lPs from Pantoea agglomerans represented by both linear and branched tetra- and pentasaccharide repeating units were described for the first time. the heterogeneity of the lPs molecule itself and the presence of several lPs in the bacterial cell, which differ in the structure of lipids a, O-specific polysaccharide chains, serological activity, as well as endotoxic proper-ties, such as toxicity and pyrogenicity, were shown. such heterogeneity represents one of the mechanisms of lPs multifunctionality. Based on the antigenicity of lPs, serotyping of P. agglomerans strains and their assignment to 10 serogroups were carried out for the first time. the high immunomodulatory activity of P. ag-glomerans lPs suggests the possibility to use their oligosaccharide fragments in the development of conju-gated vaccines against diseases caused by gram-negative bacteria.

k e y w o r d s: Рantoea agglomerans, О-specific polysaccharide, lipid a, serological activity, tlR 4 receptor, biological activity.

The representatives of the enterobacteriace-ae family, an essential part of the biosphere and widely distributed in the environment,

were among the first microorganisms that became a subject of research. Their activity affects the various spheres of human life significantly. Although en-terobacteriaceae family is the most studied (pheno-typically and genotypically) family, its classification is still changing and improving. It happens due to the considerable biological diversity of enterobacte-ria that complicates the classification of the family members. The greatest difficulties arise when iden-tifying newly discovered and poorly studied species.

However, in the recent years, the improvement to traditional and the development of new methods in systematic have resulted in the identification of 48 enterobacteriaceae genera, among them is Pan-

toea, which is related (for 25%) to the typical genus escheriсhia. This level of relationship is fairly high compared to other genera, such as Budvicia (1%), Pragia (5%) and Rahnella (15%) [1].

P. agglomerans is considered one of the most abundant microorganisms, at least in areas inhabited by humans. The ability of these bacteria to compete and survive in various environments makes them especially useful for biocontrol, bioremediation, and biodegradation. P. agglomerans representatives have been classed over the years with various bac-teria genera and types: enterobacter agglomerans, erwinia herbicola, erwinia milletiae. Only in 1989, the new genus Pantoea, which includes the P. agglo-merans species having a diverse range of properties, was applied to the enterobacteriaceae family [2]. Initially, representatives of this species were found

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in plants [1, 3], later they have been determined in water, soil, dust, air, plant and animal products, in the body of arthropods and other animals, and some-times in humans (in wounds, blood, internal organs) [1, 4, 5]. This bacterium in humans can have an am-biguous effect, both pathogenic and beneficial. On the one hand, it can cause diseases in people who in-hale organic dust [6], on the other hand, it can stimu-late the production of substances that are effective in the treatment of cancer and other diseases [7].

Recently, Nicoletti et al. suggested that P. ag-glomerans along with other 8 bacteria species (aero-monas hydrophila, Brevundimonas vesicularis, chromobacterium violaceum, citrobacter youngae, empedobacter brevis, Pseudomonas putida, Pseu-domonas stutzeri and streptococcus mitis) found in the Comano spring water can contribute to its regene rative and wound-healing properties [8].

The publications of the recent years have con-sidered the promising potential of P. agglomerans strains in fighting malaria. Bacteria of this species were detected in the midgut of anopheles mosqui-toes infected with Plasmodium [9-11]. About 300-500 million cases of malaria resulting in 1.2 million deaths are registered annually [11]. The symbiotic existence of Pantoea with malaria mosquitoes en-ables the development of a strategy called para-transgenesis, in which, bacteria express antiplas-modic effector proteins in the mosquito’s midgut. Researchers demonstrated that the introduction of P. agglomerans strains inhibited (98%) the develop-ment of the human malaria parasite Plasmodium falciparum and the rodent Plasmodium berghei [11]. These results provide new approaches to combating this lethal disease.

Lipopolysaccharides (LPS), the main compo-nents of the outer membrane of gram-negative bacte-ria, play an important role in host-pathogen interac-tions in animals and plants [12]. The outer membrane of gram-negative bacteria has a unique asymmetric phospholipid bilayer structure: its inner leaflet con-sists of glycerophospholipids, whereas the outer one is rich in lipopolysaccharide covering about 75% of the cell surface. Integral membrane proteins, porins, are also embedded in the outer membrane, and to-gether with LPS provide a barrier of permeability for different types of molecules, such as detergents, an-tibiotics, metals. The barrier properties of the outer membrane are attributed to low fluidity and a highly ordered structure of the LPS monolayer [1, 13, 14].

Although lipopolysaccharides (endotoxins) were discovered more than 100 years ago, these

complex macromolecules still draw attention not only of biochemists, but also chemists, physicists, molecular biologists who study their structure and biological activity.

One of the mechanisms that determine the li-popolysaccharide polyfunctionality could be the heterogeneity of its molecule, consisting of carbohy-drate and lipid parts, and the presence in the bacte-rial cell of several lipopolysaccharides, which differ in composition, structure and functions. Studies of lipopolysaccharides facilitate the solving such fun-damental problems as: the bacteria classification, determining the correlation between the structure and function of lipopolysaccharides, the mechanisms of the immune response, the development of sero-logical classification schemes, based on structural variations in LPS O-specific chains. Thus, the struc-tural features of LPS have been successfully used in chemotaxonomy of some gram-negative bacteria such as escherichia coli, salmonella typhimurium, shigella flexneri, Pseudomonas aeruginosa, Pseu-domonas syringae. However, in P. agglomerans taxonomy, there are still many unsolved problems. At the same time, the published data on isolation and characteristics of the lipopolysaccharides from P. agglomerans representatives remain extremely limited. Since P. agglomerans constantly enter into the gastrointestinal tract of humans and farm ani-mals with vegetables and other plant foods, study the functional and biological properties of LPS, isolated from representatives of this species, in particular, toxici ty, pyrogenicity, antigenic properties, are vi-tally important. These properties are known to de-pend, first of all, on the composition and structure of LPS molecules [15, 16].

structure and peculiarity of the structural arrangement of P. agglomerans lps. The struc-ture of LPS from P. agglomerans, like the structure of LPS from other gram-negative bacteria, include three components, namely O-specific polysaccharide (known as O-antigen) (OPS), core oligosaccharide (COS) and lipid A (Fig. 1).

The LPS molecule is anchored into the outer membrane of gram-negative bacteria via hydropho-bic interaction between lipid A fatty acids and phos-pholipids.

The complete LPS molecule, which comprises all three components, is called the smooth (S)-form LPS. The LPS, which lacks the O-specific polysac-charide and/or part of the core oligosaccharide, is called the rough (R)-form LPS (Fig. 2).

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The absence of OPS in LPS of some bacteria occurs as a result of either the absence or inactiva-tion of the O-antigen gene cluster. When bacteria are not able to polymerize the O-chain, they synthesize LPS consisting of the only one O-unit attached to COS. Several different forms of LPS can coexist in the same strain. The OPS chain varies considerably in length: from one to more than 50 O-units. The distribution of the chain length is modal (except for

bacteria that have S-LPS) and specific for different bacteria strains.

The lipid A structure is the most conserva-tive component of the LPS molecule, whereas the O-poly saccharide chains are the most variable one. Each component performs specific functions: OPS are responsible for the serological activity of bacte rial cells and serve as receptors for bacterio-phages and bacteriocins; the core oligosaccharide

Fig. 1. typical e. coli lPs (O-antigen polysaccharide, core oligosaccharide, and lipid a) arrangement in outer membrane [15]

Fig. 2. the common structure of the lPs molecule [26]

l. D. Varbanets, Т. V. Bulyhina, l. А. Pasichnyk, n. V. Zhytkevich

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is involved in maintaining the integrity of the outer membrane, and lipid A is the LPS anchor to embed in the bacterial outer membrane via electrostatic and hydrophobic interactions with phospholipids and is responsible for the endotoxic properties of gram-negative bacteria [16].

The carbohydrate part (O-specific polysaccha-ride and core oligosaccharide) is attached to lipid A by a glycosidic bond through the residue of 2-keto-3-deoxyoctonic acid (KDO) (Fig. 3) [16, 17]. This labile acid bond can be selectively cleaved under mild acid hydrolysis conditions into carbohydrate and lipid parts.

Lipid A consists of two β (1→6)-linked D-glucosamine residues, which carry fatty acids and phosphate groups attached via amide and ester bonds at the C-1 and C-4ʹ positions [18-20] (Fig. 4). Fatty acids can be saturated (in rare cases, unsatu-

rated), as well as hydroxylated. 3-OH-hydroxy acids are differentiating for certain types of bacteria. Therefore, the composition of fatty acids is used as one of the chemotaxonomic criteria in the bacterial systematics .

In lipid A of the studied P. agglomerans [21] strains, the following fatty acids were found to pre-dominate: 3-OH-C14:0 (31.7-60.6%), C14:0 (12.9-30.8%), C12:0 (8.2-30.4%), C16:1 (4.2-27.4%), C16:0 (3.3-17.3%). LPS of the two studied strains also contained 2-OH-C14:0 acid. Only one strain lacked C14:0 acid and three strains contained C18:0 acids. In some cases (3 strains), insignificant amounts of C18:0, as well as cis- and trans-C18:1 acids were de-tected; aic C15:0 acid was found in only one strain. The fatty acid composition of lipid A from studied early P. agglomerans strain was shown to distin-guish by the absence of C16:1, C18:1 and C18:0 acids

Fig. 3. structures of lipid a variants: a – hexaacyl, b – pentaacyl, c – tetraacyl, d – heptaacyl [26]

a b

c d

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[22]. The analysis of the published data indicates a variety of lipid A fatty acid compositions of LPS in different P. agglomerans strains.

The presence of only one 3-hydroxytetradeca-noic acid, which acylates amino and hydroxy groups of glucosamine residues, is a distinctive feature of lipids A of the enterobacteriaceae family members, whereas, for the majority of the studied gram-nega-tive bacteria strains, the fatty acid composition is a species-specific characteristic [23].

Thus, despite a conservative structure, lipid A is characterized by considerable microheterogeneity, which depends on various factors, including bacte-rial adaptation to changing environmental condi-tions, incomplete biosynthesis, chemical modifica-tions emerged in isolated lipids. Lipids A vary in the amount of fatty acids, their distribution along the disaccharide molecule of glucosamine, chain length and stereochemistry. The presence of charged groups in lipid A is essential for a variety of LPS biological activities, including the resistance of bacteria to an-tibiotics [24]. Thus, phosphate groups may be substi-tuted with polar or other groups. Most common polar substituents of phosphate groups, which are usually present in non-stoichiometric amounts, are: seconda-ry phosphate (lead to the formation of a diphos-phate group), hydrogen, heptose, galacturonic acid, phosphoethanolamine, and 4-amino-4-deoxy-L-arabinose [12, 25]. These substituents affect the bio-logical properties of not only LPS but also the entire bacterial cell. Thus, it was established [26, 27] that the substituents at 4ʹ-phosphate of glucosamine II result in the bacterial resistance to some polycatio-nic antibiotics, in particular, polymyxin B. In case, when the OH group at 4ʹ-phosphate of glucosamine is not substituted, polymyxin B is attached to the OH group, and such bacteria are polymyxin sensitive. When there is substituent, such as 4-amino-4-deoxy-L-arabinose, at the OH group, polymyxin is not able to bind, and such bacteria become polymyxin-re-sistant. Since only one P. agglomerans strain among 14 studied showed resistance to polymyxin B [36], it can be assumed that lipid A of LPS isolated from this strain contains substituent 4-amino-4-deoxy-L-arabinose. LPS of the rest 13 strains presumably do not contain a substituent at C4′ of glucosamine II. This assumption proved to be correct when the lipid A structures of three P. agglomerans strains 8674T,

7604, and 7969 were analyzed by GC-MS and high-resolution ESI-MS [28, 29].

Usually, lipid A has a hexaacyl structure, in which 6 acyl chains of different lengths are esterified

with glucosamine disaccharide (Fig. 3). Howe ver, certain bacteria can synthesize under-acylated li-pid A, for example, tetra- or pentaacyl forms [30, 31] or even over-acylated, such as the heptaacyl lipid A [31, 32].

Tsukioka et al. showed that lipopolysaccharide of P. agglomerans carries at least two types of li-pids A with different levels of acylation [22]. One was the same as escherichia coli 4′-monophos-phorylhexaacyl-lipid A, and the other was similar to salmonella enterica 4′-monophosphorylheptaacyl-li-pid A. These LPS also differed in molecular weight: 5 kDa and 30-60 kDa, respectively.

Similar results were obtained by other resear-chers who detected two lipid A types in the LPS from P. agglomerans [33, 34]. They are characteri-zed by low molecular weight and differ from the previously described structures only by the presen-ce of an additional oxygen atom at the acyloxyacyl ester-linked side-chain of the distal portion of the molecule.

GC-MS and high-resolution ESI-MS analysis of lipids A of P. agglomerans 7604, 7969 and 8674T

strains revealed their high heterogeneity [28, 29]. Lipid A from P. agglomerans 8674T was represen-ted by hexa-, penta-and tetraacyl species [29]. The hexa acyl species included four 3-OH-C14:0 residues, one C14:0 residue and one C12:0 residue; the pen-taacyl species was characterized by the absence of one C14:0 residue and the tetraacyl species did not contain two fatty acid residues, namely 3-OH -C14:0 and C14:0. In addition, peaks corresponding to hexa-, penta- and tetraacyl species, in which C14:0 or C12:0 residues were substituted with C16:1 or C18:1 resi-due, respectively, were observed. Moreover, in the hexaacyl species, additional peaks appeared, probab-ly due to the substitution of C14:0 for C16:0 or 2-OH-C14:0, as well as corresponding monophosphoryl-ated derivatives, were registered.

Analysis of the mass spectra of the lipid A of LPS from P. agglomerans 7969 showed two main peaks for diphosphorylated disaccharides, which correspond to hexa- and tetraacyl species, with the same residues as P. agglomerans 8674T strain. These data are consistent with previously published data on the structure of lipid A of P. agglomerans [22]. There are also peaks corresponding to two more hexaacylated species, in which C14:0 acids are likely to be replaced by C16:0 or 2-OH-C14:0; and peaks corresponding to monophosphorylated derivatives.

On the spectrum of P. agglomerans strain 7604, two species of lipid A were observed: pentaacyl with


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four 3-OH-C14:0 residues and one C12:0 residue and tetraacyl - without one 3-OH-C14:0 residue [29]. Both derivatives demonstrated peaks appeared as a result of substitution C12:0 with C18:1, and peaks corresponding to monophosphorylated derivatives.

It can be concluded based on the obtained data on LPS lipid A structures of three P. agglomerans strains that they are heterogeneous, characterized by different degrees of acylation and do not contain any monosaccharides as substitutes at C4′ position. These findings were confirmed by the results of the studying the sensitivity of these strains to polymy-xin B [35].

OPS structure. It is known that OPS structure is characterized by variability [26]. To determine OPS structure, researchers use the traditional methods , such as gas-liquid chromatography, periodate oxida-tion, methylation, determination of the absolute con-figuration of monosaccharides along with the most advanced ones, such as chromatography-mass spec-trometry, NMR spectrometry, computer analysis [36]. The use of a combination of modern biochemi-cal and biophysical methods enabled to characterize the unique structures of the OPS in many bacteria species [26, 37].

Typical components of OPS are monosaccha-rides, which occur in nature. Most of them exist in the form of pyranose, but some can be represented by furanoses (arabinose, ribose, xylose) or by both forms (galactose, fucose, paratose). In some OPS, ribose occurs in the form of pyranose, and N-acetyl-galactosamine – in the form of furanose. An OPS usually contains N-acetyl and O-acetyl groups, less often – methyl group bound to hydroxyl or amino groups. In various OPS, the hexuronic acid may occur as an amide with an amino component, such as 2-amino-2-deoxyglycerol (GroN) or amino acid (L-lysine and its Nε-(1-carboxyethyl) derivative). Phosphate was found only in the form of a diester, including cyclic phosphate [26].

The O-antigen structures are mostly established for bacteria from the enterobacteriaceae family. Thus, at present, the full O-antigen structures have been identified for more than 180 strains of e. coli,

which is a type species of the enterobacteriaceae family. They contain linear or branched tri-, tetra-, penta- and hexasaccharides; less commonly disac-charides or homopolysaccharides [26].

Data on the OPS structures of P. agglome-rans representatives are limited. Thus, it was shown [34] that the OPS from P. agglomerans contain a branched heteropolymer with repeating pentasac-charide units, containing rhamnose, glucose, NAc-glucosamine and NAc-fucosamine residues (I).

A neutral OPS isolated from P. agglomerans strain FL1 was found to have a completely different structure. Thus, it is constituted by a homopolymer arranged as linear tetrasaccharide repeating units consisting of D-rhamnose residues [38]:→2)-α-D-Rhap-(1→2)-β-D-Rhap-(1→3)-α-D-Rhap-(1→2)-α-D-Rhap-(1→

Hashimoto et al. showed that the structure of OPS of LPS from P. agglomerans IG1 (IP-PA1) is composed of linear tetrasaccharide repeating units consisting of glucose and rhamnose residues, in which 40% of one of the rhamnose residues is sub-stituted with glucose [39]:→2)-α-L-Rhap-(1→6)-α-D-Glcp-(1→2)-[β-D-Glcp-(1→3)]0.4-α-L-Rhap-(1→2)-α-L-Rhap-(1→

This means that if OPS consists, for example, of 10 repeating units, only in four of them rhamnose is replaced by glucose, and the rest are unsubstitu-ted. This leads to one of the possible heterogeneities of the LPS molecule that plays a significant role in its biological activity.

A similar structure, where rhamnose residues were substituted with glucose, was identified in the OPS from shigella flexneri serotype X [40]:→3)-β-D-GlcpNAc-(1→2)-[β-D-Glcp-(1→3)]-α-L-Rhap-(1→

The structures of the OPS from P. agglome rans 8674T and P. agglomerans 7604 were shown to have similar features and constituted by branched pen-tasaccharides containing 3 rhamnose residues and 2 glucose residues, one of which was in the late ral position (II). The only difference was that in 8674T

[→3)-α-L-FucpNAc-(1→3)-α-L-FucpNAc-(1→3)-β-D-GlcpNAc-(1→]6↑ (I)1

α-D-Glcp-(1→2)-α-L-Rhap

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strain the glucose residues in the lateral position had β-configuration, and in 7604 strain - α-configuration [29].

It was reported that the main chain of neutral OPS isolated from serratia marcescens 111 LPS (O29) has the same structural features; however, the lateral substituents were not identified in its struc-ture [41].

The structure of OPS from P. agglomerans 7969 [28] differ greatly from the structures described above and is constituted by a linear tetrasaccharide consisting of rhamnose, mannose, fucose, and N-acetylglucosamine residues (III).

Its structure appeared to be heterogeneous due to non-stoichiometric (~25%) substitution at posi-tion 6 of N-acetylglucosamine residue with glycerol 1-phosphate. Earlier, this type of heterogeneity was identified in the OPS from Proteus vulgaris TG103 [42]. There has been no reported data on such struc-ture so far. However, the rhamnose and glucosa-mine disaccharide fragments are quite common in gram-negative bacteria, for example, agrobacterium tumefaciens F1, serratia marcescens O17 and O19, klebsiella pneumonia O12. In OPS from steno-trophomonas maltophilia O6, this disaccharide is xylosylated at position 4 GlcNAc [43].

lps functions. The structural components of the LPS molecule differ not only in structure but also in functions and biological activity. Thus, lipid A is responsible for the endotoxic activity of the molecule, while the O-specific polysaccharide is associa ted with the O-serological specificity of the bacterial cell. Therefore, subtle variations in LPS structures are the basis for the development of intraspecies serological classification schemes for gram-negative bacteria. The first serological classi-fication scheme based on the structures of O-speci-

fic polysaccharides (10 structures) was proposed by Kauffmann and White for the salmonella genus [44]. The authors showed that the serotypes of salmonella genus members are determined by the presen ce of substituents at mannose or galactose. Thus, 3,6-dide-oxy-hexoses, such as paratose (s. paratyphi), abe-quose (s. abortus equi, s. typhimurium), tyvelose (s. typhi, s. trasbourg) can be bound to the man-nose as the side chain via the α-1,3 bond. Glucose attached to galactose by α-1,4 or α-1,6 bonds may be present as another side chain. In polysaccharides of group B salmonella, abequose is often acetyla-ted at C-2. Thus, in s. typhi murium, most abequose residues were found to be present in the form of O-acetyl derivatives.

Significant progress made in the last decades in studying OPS structures has allowed determining 46 serogroups for salmonella enterica, 46 – for shigella representatives, 76 – for Proteus, 61 – for Providen-cia, 39 – hafnia. The most large-scale study has been carried out on e. coli, in which more than 180 serogroups have been identified [45].

A study of the serological activity of LPS from 14 strains of P. agglomerans by Ouchterlony double immunodiffusion between antisera to the studied strains and LPS isolated from the homologous strains demonstrated that LPS exhibit antigen activity [21]. However, it was found in serological cross-reaction tests between antisera and LPS from different strains that only some of them exert cross-reactivity. These findings enabled to divide, for the first time, the 14 studied P. agglomerans strains into 10 serogroups based on the LPS O-antigenic properties. This is the only study on the serological identification of P. ag-glomerans strains known currently [21].

lps biological activity. The complex structure of the LPS molecule and its heterogeneity, as well as

P. agglomerans 8674т

→2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→2)-α- D-Glcp-(1→β-D-Glcp-(1→3)

(II)P. agglomerans 7604

→2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→2)-α- D-Glcp-(1→ α-D-Glcp-(1→3)

→3)-α-L-Rhap-(1→6)-α-D-Manp-(1→3)-α-D-Fucp-(1→3)-β-D-GlcpNAc-(1→ (III)Gro-1-P-(O→6)


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the presence of several LPS differing in molecular mass and structure within the same strain of bacte-ria, determine LPS polyfunctionality. On the one hand, LPS perform a biological protective function, are involved in cell adhesion, exhibit mitogenic ac-tivity and antitumor effect, and can serve as markers for bacterial strain identification. LPS can activate B- and T-lymphocytes, granulocytes and mononu-clear cells and thus, are considered as potential im-munomodulators. LPS, having antigenic activity, serve as biological recognition components, carriers of specific information determining the relation-ship between bacteria and macro-/microorganisms. On the other hand, as bacterial endoto xins, LPS in higher animal and human induce a wide range of pathophysiological reactions that can lead to septic shock, and be fatal in some cases. Owing to these properties, LPS contribute to the pathogenic poten-tial of gram-negative bacteria, and thus, to the out-break of severe infectious diseases. The pathogenesis of sepsis includes the following phases: neutrophil, monocyte and macrophage inflammatory reactions, intravascular coagulopathy, endothelial cell injury, and hypotension. Thus, LPS has a pleiotropic effect on tissues and organs that can lead to death [46]. Re-cently, researchers have found specific LPS recep-tors on the immune cell surface and blood proteins, which can recognize the minor differences in the LPS structure and make an organism to respond. Since the biological activities of LPS correlate with their structures, the latest findings on the LPS struc-tures have enabled to investigate the mechanism of the LPS biological activity at the molecular and cel-lular level. Understanding these mechanisms is es-sential for solving some fundamental problems of immunogenesis and cell reception. The crucial point in the investigation was the discovery by Bruce Beutler in 1998 of the LPS receptor, Toll-like recep-tor, when he studied the effect of LPS on mice [47]. This receptor is similar to the protein coded by the Toll gene identified in Drosophila in 1995. LPS was shown to trigger an immune response via interac-tion with Toll-like receptor 4 (TLR4), which is a key receptor of innate immunity.

Identification of the LPS receptor was essen-tial for understanding the mechanisms of innate immunity. In 2011, Bruce Beutler, Jules Hoffmann and Ralph Steinman received Nobel Prize in Physio-logy or Medicine for their discoveries of one of the molecular mechanisms for activation of innate im-munity [48]. Toll-like receptor activation occurs

when it binds to ligands, which can be certain moie-ties of bacteria, fungi or viruses. Fig. 4 presents the scheme, in which LPS of gram-negative bacteria is a ligand, and Toll-like receptor 4 (TLR4), which is a surface protein remotely related to the IL-1 recep-tor [48]. For the recognition of lipopolysaccharide, TLR4 require the presence of transport proteins such as lipopolysaccharide binding protein (LBP), soluble and membrane-bound CD14 proteins [49]. Genetic and biochemical analyses revealed that for the subsequent recognition of the LPS/CD14 com-plex by the TLR4 receptor, other proteins are also needed, in particular, TLR4-associated co-receptor MD-2 [50]. This protein belongs to the family of lipid-binding proteins [51, 52], which characterized by the β-sandwich structure. In the signaling path-way, TLR4 recognizes hexaacyl diphosphorylated domain of lipid A. The binding to LPS is achieved by intercalation of lipid A acyl chains into hydropho-bic region of β-sandwich [53, 54]. This model was confirmed by structure analysis of MD-2 associated with lipid IVa, as well as a TLR4/MD-2 heterodimer in complex with the structurally related to lipid IVa synthetic compound E5564 [55, 56]. Activated by this complex macrophages or mononuclear phago-cytes trigger the biosynthesis of various inflamma-tory mediators, such as TNF, IL-1, IL-6, IL-8, as well as adhesive molecules necessary for the adap-tive immune response [57, 58].

A leading role in the LPS induction of most biological effects is performed by lipid A [58], be-cause after removal of the core oligosaccharide and the O-specific polysaccharide from LPS, TLR4 is still able to recognize it. Tetraacyl and hexaacyl LPS from e. coli are the most studied. The cumulative effect of acyl groups in lipid A has been revealed: the more acyl residues and the longer the chains of acyl groups in lipid A structure, the more proinflamma-tory cytokines macrophages produce. Thus, a direct link between the biological activity of LPS and the chemical structure of lipid A was demonstrated.

The first data on the biological activity of LPS extracted from 10 strains of P. agglomerans isolated from dust, air, human and animal sources were pre-sented by Dutkiewicz in 1976 [59]. Thomas in 1956 showed that P. agglomerans LPS mixed with adren-aline caused dermal hemorrhagic lesions in rabbits and death of the 10-day-old chicken embryos [60].

In further research, a significant increase in the number of polymorphonuclear leukocytes and al-veolar macrophages was detected 24 h after guinea

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pigs inhaled live cell suspensions or LPS from P. ag-glomerans isolated from cotton [61-63]. These LPS also caused the death of mice and were pyrogenic for rabbits [64].

In 1991, the compound, which activated mac-rophages after oral or subcutaneous administra-tion, was discovered in an aqueous extract of wheat flour [56]. The active compound was identified as LPS isolated from P. agglomerans cells that were in symbiosis with wheat. The authors called this li-popolysaccharide as IP-PA1 (immunopotential from P. agglomerans, a former name: LPSp) and showed its effectiveness in various areas, such as healthy diet (to prevent or reduce metabolic syndrome), skin care products (to maintain healthy skin), as well as active ingredients in animal feed and aquaculture. Later, IP-PA1 administered orally or subcutaneously was shown to activate peritoneal macrophages and increase the activity of phagocytes through TLR4 signaling pathway [55, 56].

Iwamoto et al. demonstrated that intravenous co-administration of LPS from P. agglomerans with cyclophosphamide resulted in complete regression of mouse hepatoma MH134 [65]. In mice treated by this method, the researchers observed necrosis and inflammatory cell infiltrates (CD4+ and CD8+ T-lymphocytes, macrophages, neutrophils) in tu-

Fig. 4. tlR4 receptor recognition of lipid a [61]

Induction of formationTNF, IL-1, IL-6, IL-8

(LPS-MD-2-TLR4)2

(pCD14-LPS)2

LBP-LPS

mCD14-LPS

LPS

gram-negativebacteria

mors and the related increase in serum cytokines (TNF-α, IL-1α, IL-6, G-CSF (granulocyte colonies stimulating factor)). It was concluded that treatment with LPS was effective mainly due to increased T-cell mediated antitumor immunity developed by the synthesis of TNF-α and other cytokines. These re-sults were confirmed by Inagawa et al. [64, 66] who showed the complete regression of syndromal mouse tumors, Meth A fibrosarcoma, MH134 hepatoma, and Lewis carcinoma after combined treatment with intravenous administration of LPS with cyclophos-phamide. The researchers indicated that the induc-tion of TNF-α plays an important role in successful therapy and that subcutaneous drug administration was low toxic (by 230-380 times less) compared to intravenous administration.

Japanese researchers described this new medi-cation, containing low molecular weight (5 kDa) LPS and with no side effect, as immunostimulator P. ag-glomerans 1 and suggested it for the treatment of a range of diseases including malignant tumors by oral or intravenous administration [47].

In addition, oral administration of IP-PA1 was shown to prevent diabetes, as well as infectious disea ses caused by bacteria and viruses [56]. Howe-ver, it is still not clear whether IP-PA1 administered orally, and absorbed in the intestine, enters into tis-


14


sue macrophages. From drug delivery point of view, it is important to identify the IP-PA1-binding cells after IP-PA1 oral administration.

Dutkiewicz et al. reported that significant amounts of P. agglomerans were found in grain and flour dust in the form of globular nanoparticles measuring 10-50 nm, which can be described as “endotoxin super macromolecules” [67]. Their con-centration in dust was found to range from 104 to 105 CFU/g and formed 73.2-96% of the total gram-negative bacteria in the air [67]. Therefore, resear-chers consider P. agglomerans as major causative agents of toxic pneumonia in agricultural workers. Indeed, P. agglomerans endotoxins were shown to cause inflammatory and fibrotic changes in lungs, to stimulate alveolar macrophages, to produce anion superoxide, interleukin-1 and chemotactic factors for other macrophages and neutrophils, and to increase the concentrations of pulmonary toll-like receptors and chemokines [67]. Researchers believe that LPS and proteins from P. agglomerans should be con-sidered as major causative agents of occupational diseases, such as allergic dermatitis in farmers, and also allergic pulmonary disorders in cattle not only in the cotton industry but also for the grain industry and agriculture.

In 2016, researchers obtained impressive results demonstrating that the pretreatment with LPS from P. agglomerans (10 ng/ml) or with monophosphoryl lipid A for 18 h was effective in the prevention of Alzheimer’s disease by enhancing phagocytosis of β amyloid by brain microglia [68]. Microglial cells were isolated from adult mouse brain.

Thus, the above results, indicating a high im-munomodulatory activity of LPS, suggest their usa-ge in the development of new drugs for various ap-plications. However, therapeutic utilization of LPS is impeded by their high toxicity and pyrogenicity, as well as the insufficiency or inconstancy of their stimulating effect [69].

With regard to P. agglomerans, the LPS from the studied strains were characterized by a relatively low toxic effect (LD50 420 and 147 µg/mouse), in con-trast to LPS from other enterobacteriaceae fami ly members (LD50 range from 3.6 to 75 µg/mouse) [70]; however they induce pyrogenic reactions [70].

Since lipid A is responsible for endotoxic ac-tivity, the researchers studied the role of fatty acids, in particular, their length and distribution on glu-cosamine molecules. It was found that lipids A with short-chain fatty acids were less toxic, or completely

non-toxic compared to lipids A with long-chain fatty acids [14, 71, 72]. Hexaacyl bisphosphorylated lipid A structure with an asymmetric (4+2) distribution of acyl chains attached to diglucosamine was shown to be the most active, while its various modifications significantly affect the biological activity of LPS. Such hexaacyl bisphosphorylated lipid A is charac-terized by a large tilt angle, a conical (hexagonal) molecular shape and high endotoxic activity. Hexa-acyl monophosphorylated lipid A has a smaller tilt angle, and the conical shape is less expressed tending to be more cylindrical (cubic) shape. This correlates with less pronounced endotoxic activity. Penta- and tetraacyl lipid A or hexaacyl lipid A with symmetric acyl chain distribution (3/3) are characterized by a small tilt angle, a cylindrical (lamellar) shape and are endotoxically inactive, but can be antagonistic (Table) [73].

Thus, it was shown that lipid A conformation plays a significant role in the manifestation of the LPS biological activity. Thus, so-called endotoxi-cally active conformation corresponds to the cubic and hexagonal supramolecular structures, while the lamellar structure is endotoxically inactive.

Although lipid A was found to play the main role in the manifestation of the LPS biological activi-ty, the length of the OPS, as well as the molecular mass of LPS, is also important [22, 74-76]. Thus, the literature described the LPS structure from one of the P. agglomerans strains, which contained at least two types of lipids A with different level of acylation. One of them is similar to 4′-monophos-phorylhexaacyl lipid A from escherichia coli, and the other – to 4′-monophosphorylheptaacyl lipid A from salmonella enterica. These LPS also dif-fered in molecular weight: low 5 kDa and high 30-60 kDa, respectively. Hexaacyl lipid A was found to have high activity, including endogenous induction of TNF, while heptaacyl lipid A had lower activity. These LPS from P. agglomerans exhibited benefi-cial therapeutic effects, which were not observed for other LPS. It was suggested that these effects could not be attributed solely to the structure of lipid A. The length of the OPS chain and the LPS compo-sition may also contribute greatly to the biological activity of lipid A.

Thus, Kohchi et al. [7] found that the OPS chain of IP-PA1 from P. agglomerans is shorter and charac terized by a much lower molecular weight than the LPS of the type e. coli O:113 strain. There-fore, LPS IP-PA1 easily forms small-diameter mi-

15

celles, which can easily cross the mucous membrane and/or skin, enhancing positive effect of treatment after oral or subcutaneous administration.

Pupo et al. [77] demonstrated that LPS from e. coli containing short and long polysaccharide chains exhibited various biological activities on human macrophages. An essential role in the in-teraction of S-LPS with host cells is played by the LPS-binding protein. These results suggest that the polysaccharide part may affect the biological activity of LPS through serum factor.

Kadowaki et al. [78] divided the LPS IP-PA1 into two fractions: high-molecular (30-60 kDa) and low-molecular (5 kDa). The first fraction contains long-chain OPS and the last one contains short oli-gosaccharides. These fractions induced different NO production in RAW264.7 cells.

The described results support the involvement of the LPS polysaccharide region in its biological ef-fects.

Thus, the reported results [59, 62, 63, 78] indi-cated that the potential of biological activity of the LPS preparations from P. agglomerans was very similar to that of the LPS from most enterobacte-riaceae species (for example, citrobacter freundii, enterobacter aerogenes, enterobacter cloacae, klebsiella oxytoca, salmonella typhi, salmonella typhimurium) or Pseudomonadaceae (for example, Pseudomonas putida), and was higher compared to

chemical composition, molecular conformation and biological activity of different lipids a

the LPS from other gram-negative bacteria, such as agrobacterium sp. and Xanthomonas sinensis [62, 63] or acinetobacter calcoaceticus and alcali-genes fecalis [79].

The data found in literature and our own experi mental results indicate the importance of the study of lipopolysaccharides of gram-negative bac-teria, including P. agglomerans, a poorly-studied member of the enterobacteriaceae family. Discove-ry of the heterogeneity of LPS, distinguished by unique structures of O-specific polysaccharides, lipids A, charac terized by different degrees of acylation, as well as serological activity and endo-toxic properties not only contribute to the biological charac teristics of the species but also could stimulate the development on their base of new drugs of vari-ous applications.

Further research focused on the clarification of the following aspects could be promising:

- the role of LPS in the pathogenesis of infec-tious diseases - this will contribute to the develop-ment of a new effective approach to combating disea ses caused by gram-negative bacteria;

- the ability of modified lipopolysaccharides to modulate both the innate and adaptive immune re-sponse;

- the possibility of using synthetic polysaccha-rides as components of vaccines, adjuvants, antican-cer drugs, nonspecific stimulants.

Conformation Supramolecular structure

Fatty acid composition Toxicity Species of bacteria

Lamellar

3-OH-C14:0 (2)3-OH-C10:0 (2) C12:1 (1) Not toxic

Rhodobacter capsulatus

3-OH-C14:0 (2)3-OH-C16:0 (2) Rhodospirillum fulvum

Cubic 3-OH-C14:0 (2) C12:1 (2)

Moderately toxic escherichia coli

Hexagonal

3-OH-C10:0

Hight toxic

Rhodocyclus gelatinosus

3-OH-C14:0 (2) C12:0 C16:0

salmonella minnesota


16


Conflict of interest. Authors have completed the Unified Conflicts of Interest form at http://ukrbio chemjournal.org/wp-content/uploads/2018/12/coi_disclosure.pdf and declare no conf lict of interest .

ЛіпопоЛісахариди Рantoea agglomerans: структура, функціонаЛьна та біоЛогічна активність

Л. Д. Варбанець, Т. В. Булигіна, Л. А. Пасічник, Н. В. Житкевич

Інститут мікробіології і вірусології ім. Д. К. Заболотного НАН України, Київ;

e-mail: [email protected]

В огляді наведені дані літератури, а та-кож результати власних експерименталь-них досліджень ліпополісахаридів (ЛПС) грамнегативних бактерій. Основну ува-гу автори приділяють Рantoea agglomerans, представнику родини enterobacteriaceae. Вперше описано унікальні структури О-специфічних полісахаридних ланцюгів їхніх ліпополісахаридів, які можуть бути як розгалу-женими, так і лінійними тетра- і пентасахарид-ними ланцюгами, що повторюються. Показана гетерогенність як самої молекули ЛПС, так і присутність в бактеріальній клітині декількох ЛПС, які відрізняються структурою ліпідів А, О-специфічних полісахаридних ланцюгів, серологічною активністю, а також ендоток-сичними властивостями, зокрема токсичністю і пірогенністю. Така гетерогенність є одним із механізмів поліфункціональності ЛПС. На основі О-антигенності ЛПС вперше було проведено се-ротипування штамів Р. agglomerans і віднесено їх до 10 серогруп. Висока імуномодулювальна активність ЛПС Р. agglomerans дозволяє при-пустити використання їхніх олігосахаридних фрагментів для створення кон’югованих вакцин проти захворювань, спричинених грамнегатив-ними бактеріями.

К л ю ч о в і с л о в а: Рantoea agglomerans, О-специфічний полісахарид, ліпід А, серологічна активність, TLR 4 рецептор, біологічна активність.

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